Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs that are highly expressed in KS lesions and KSHV- associated lymphoproliferative malignancies. Ectopic expression and miRNA inhibition approaches, combined with gene expression profiling, have identified target mRNAs that are involved in the regulation of critical biological processes such as angiogenesis, apoptosis, endothelial cell differentiation, and immune surveillance. In addition, several miRNAs may regulate latency by targeting viral mRNAs. Preliminary analyses of the mRNAs targeted by KSHV miRNAs in PEL cells using HITS-CLIP technology to enrich RISC-associated RNAs have identified hundreds of new mRNA targets and confirmed previously identified targets. Moreover, our analysis showed that a high percentage of RISC complexes within PEL cells contain KSHV- encoded miRNAs. Based on these data, we hypothesize that the KSHV miRNAs cause significant and tissue-specific changes in gene expression that contribute to pathogenesis and tumorigenesis. To directly address this hypothesis we propose to 1) Comprehensively identify KSHV miRNA targets by HITS-CLIP in KSHV-infected PEL and endothelial cells; 2) Characterize in vitro phenotypes and their associated regulatory pathways with respect to viral replication, the establishment and maintenance of latency, cell cycle control and resistance to apoptosis by using KSHV mutants lacking single or multiple miRNAs. In summary, combining ribonomics approaches for miRNA target identification with viral genetics, and the ability to determine viral phenotypes in cultured cells, will provide a detailed analysis of KSHV-encoded miRNA functions and may lead to the development of antiviral/antitumor strategies.